Week 2

Since I previously posted, there has been another small change to my project. The goal of creating and testing a stable cell line is still the same, as are the procedures, but a different plasmid will be used. The new plasmid is YFP-ER-E2F1. Like the previous plasmid, it still has the yellow fluorescent protein and the gene of interest for this experiment, E2F1. However, the gene that controls the “on-demand” mechanism is now ER, an estrogen receptor. In an environment where estrogen is absent, the receptor will prevent E2F1 from entering the nucleus. Since E2F1 is a transcriptional factor (a gene that effects the expression of other genes), it is in a sense inactivated by being kept out in the cytoplasm. When estrogen is present, E2F1 will enter the nucleus and its effects can be observed in the cell.

This week, we received some confusing data while running our first restriction enzyme digest. The purpose of performing the digest was simple: we wanted to double check that the enzymes would make the right cuts before we made a large volume sample to use in NEON electroporation. When we ran the products of our digest on a gel, we saw four bands. This was peculiar, because when we put the sequence for the gel into a restriction enzyme digest calculator, the results indicated that we should have seen only two bands. Eventually, we managed to trace the extra bands back to the fact that one of our enzymes was very inefficient. It left enough uncut, supercoiled, and nicked DNA to cause extra bands to appear. After determining the cause of our problem, we have ordered a new enzyme, and plan to perform another test run with it on Monday.